Proteinuria and albuminuria

Ron T. GansevoortRon T. Gansevoort

Coordinator PREVEND StudyCoordinator PREVEND StudyDepartment of NephrologyDepartment of NephrologyUniversity Medical CenterUniversity Medical Center

GroningenGroningenThe NetherlandsThe Netherlands

Ron T. GansevoortRon T. Gansevoort

Coordinator PREVEND StudyCoordinator PREVEND StudyDepartment of NephrologyDepartment of NephrologyUniversity Medical CenterUniversity Medical Center

GroningenGroningenThe NetherlandsThe Netherlands

Assessing albuminuriaAssessing albuminuria

Methodological considerations with clinical impactMethodological considerations with clinical impact

IntroductionIntroduction

Albuminuria is a urinary biomarker that has been shown to be a predictor of renal and CV events.

As such albuminuria has a place in clinical practice: kDOQI stages 1 and 2 are defined by presence of micro-albuminuria.

There is strong lobby for standardisation of measurement serum creatinine (Cleveland Clinic / IDMS traceable) to obtain the most reliable GFR estimate.

Untill recently little attention has been paid to standardisation of albuminuria (exception Miller et al, Clin Chem 2009;55:24-38)

Micro-albuminuriaMicro-albuminuria- - Definition and classification -Definition and classification -

Spot urines(first morning void, or random)

17 - 17025 - 250

< 17< 25

Alb/creat ratio

(mg/gram)

> 200> 300> 200Macro-albuminuria

20 – 20030 – 300MF

20 – 200Micro-albuminuria

< 20< 30MF

< 20Normal

AlbuminExcretion

(g/min)

AlbuminExcretion(mg/24h)

AlbuminConcentration

(mg/l)

Overnight (timed)24h urine

> 170> 250

MF

Assessment of albuminuriaAssessment of albuminuriaQuestions to addressQuestions to address

1. What assay to use? (answer: immunochemistry polyclonal)

2. What urine sample to use?

24hr urine collection, first morning void or a spot sample?

3. Which albuminuria measure to use: urinary albumin concentration, albumin/creatinine ratio, or 24hr albumin excretion?

4. Does it matter whether we use fresh urine samples or stored samples?

5. If we are going to use frozen urine samples, what is important?

pre-storage handling, storage temperature, sample handling

Monomer

Bakker , Gansevoort et al, Curr Hypert Rep 2009;11:111-7

Dimerization

Polymerization

Fragmentation

Loss of immunoreactivity ??

Type of assay

Monoclonal AB

Polyclonal AB

Colorimetric test strips

Immunochemistry based

Size exclusion (HPLC)

Assessment of albuminuriaAssessment of albuminuriaWhich assay to use?Which assay to use?

24-hour FMV Spot (morning)

Alb

umin

con

cent

ratio

n (m

g/L)

0

20

40

60

80

Median 24-hour [IQ-range] 7.6 [4.8-12.7]

Median Overnight [IQ-range] 7.2 [4.5-12.0]

Median Spot (morning) [IQ-range] 11.9 [7.8-25.8]

P = < 0.01

P = < 0.01

P = 0.43

N=250

Witte et al, JASN 2009;20:436-43

What urine samples to use ?What urine samples to use ?Median urinary albumin concentrationMedian urinary albumin concentration

PREVENDPREVEND

Urinary Albumin Concentration


Intr

a-su

bjec

t co

effic

ient

of

Var

iatio

n (%

)

0

20

40

60

80

100P = < 0.01

P = < 0.01

P = 0.08

PREVENDPREVEND

What urine samples to use ?What urine samples to use ?Coefficient of variationCoefficient of variation

N=250



Alb

umin

e:cr

eatin

ine

ratio

(m

g/m

mol

)

0

2

4

6

8

10

Median 24-hour [IQ-range] 1.00 [0.65-1.54]

Median Overnight [IQ-range] 0.67 [0.50-1.17]

Median Spot (morning) [IQ-range] 1.21 [0.68-2.37]

P = 0.023

P = < 0.001

P < 0.001


What urine samples to use ?What urine samples to use ?Median albumin:creatinine ratioMedian albumin:creatinine ratio

PREVENDPREVEND

N=250

PREVENDPREVEND

What urine samples to use ?What urine samples to use ?Coefficient of variationCoefficient of variation

Albumin:creatinine ratio

24-hour FMV Spot (morning) 0

20

40

60

80

100 P = < 0.01

P = < 0.01

P = 0.58

N=250

Intr

a-su

bjec

t co

effic

ient

of

Var

iatio

n (%

)


0

10

20

30

Overall

Male

Female

Pre

vale

nce

of

mic

roa

lbu

min

uri

a (

%)

#*

#*

#*#*

#*

#*

24hr

UAE

UACACR

0

10

20

30

Overall

Male

Female

Pre

vale

nce

of

mic

roa

lbu

min

uri

a (

%)

First Morning Void

#*

#*

#*#*

#*

#*

PREVENDPREVEND

Spot Morning Urine Sample


UACACR

What urine samples to use ?What urine samples to use ?Prevalence of microalbuminuriaPrevalence of microalbuminuria

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Which albuminuria measure to use? Which albuminuria measure to use? Predicting CV outcomePredicting CV outcome

24 hr urine First morning void

UAE (mg/24hr) UAC (mmol/L ACR (mg/mmol)

Overall 0.65 0.62 0.66*

Subgroups Male 0.64 0.62 0.68*

Female 0.66 0.59# 0.66*

<47 yr 0.58 0.52 0.52

>47yr 0.65 0.64 0.64

* p < 0.05 vs UAC, # p < 0.05 vs UAE

AUC ROC curveAUC ROC curve

N=3432

Random sample of the general populationLambers-Heerspink et al, Am J Epidemiol 08;168:897-905

Lambers Heerspink et al, submitted

N=701

RENAAL: DM2 nephropathy

Which albuminuria measure to use? Which albuminuria measure to use? Predicting renal outcomePredicting renal outcome

100 80 60 40 20 00

20

40

60

80

100S

ensi

tivi

ty (

%)

Specificity (%)

UAE 24hr; AUC = 0.78

UPE 24hr; AUC = 0.78

p<0.001

ACR FMV; AUC = 0.82

First morning void

-60

-40

-20

0

20

40

60

80

35 45 55 65 75

Age (years)

Diff

ere

nce

(%

)

UAC

UCrC

ACR

24-hour urine collection

-60

-40

-20

0

20

40

60

80

35 45 55 65 75

Age (years)

Diff

ere

nce

(%

)

UAE

UCrE

ACR

Lambers-Heerspink, Gansevoort et al, Am J Epidemiol 2008;168:897-905

PREVENDPREVEND

Which albuminuria measure to use? Which albuminuria measure to use? ACR “incorporates” the influence of ageACR “incorporates” the influence of age

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Frozen storage (-20 C) of urine samplesFrozen storage (-20 C) of urine samplesInfluence of duration of storage and sample handlingInfluence of duration of storage and sample handling

3 to 5 5 to 8 8 to 12 12 to 18 18 to 24 months

* Not significantly different from zeroHand-inversion

* *

Per

cen

tag

e ch

an

ge

in U

AC

, %

Vortex mixing

No sample handling

-80

-60

-40

-20

0

20

40

Brinkman et al, Clin Chem 2005;51:2181-3

Predictive value of albuminuria Predictive value of albuminuria Does it matter when urine has been stored frozen ?Does it matter when urine has been stored frozen ?

Brinkman et al, Clin Chem 2007;53:153-4

PREVENDPREVEND

P<0.01

Predictive value of UAE for CV endpoints

PREVENDPREVEND

Frozen storage of urine samplesFrozen storage of urine samplesDoes urinary pH matter ?Does urinary pH matter ?

-100

-75

-50

-25

0

25

50

75C

han

ge

in u

rin

ary

alb

um

in c

on

cen

trat

ion

(%

)

-20°C - 20CpH8

-80C -80CpH8

Storage condition

-

-

-

-

-

Lambers Heerspink et al, Diabetic Med 2009;26:556-9

Screening for albuminuriaThe past (1892)

Gansevoort and Ritz, Nephrol Dial Transplant 2008

ConclusionsConclusions

When assessing the clinical impact of urinary biomarkers it isessential to take into consideration methodological issues

1. Which assay was used? Polyclonal? Intra- and interassay CV?

2. What urine samples were used? Preferably 24hr collections or first morning voids

3. In case first morning void samples are used, normalise for creatine concentration

4. Fresh or frozen? Preferably use fresh urine samples.

5. If frozen, what were storage conditions and how was sample handling? Frozen at -80 0Celsius, pH adjustment (or protease inhibitors?), vortexing?

Documents

Proteinuria and albuminuria